When murine interleukin-6 is overexpressed in Escherichia coli, a small population of molecules exhibits a novel C-terminal modification. Peptide mapping, electrospray ionization-mass spectrometry, and automated N- and C-terminal sequencing identified a peptide ("tag" peptide), -Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH, encoded by a small metabolically stable RNA of E. coli (10Sa RNA) attached to truncated C termini of the recombinant protein. A mutant strain of E. coli in which the chromosomal 10Sa RNA gene (ssrA) is disrupted does not produce this C-terminal modification, confirming that the tag peptide originates from the ssrA gene.

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Protein kinases play pivotal roles in the control of many cellular processes. In a search for protein kinases expressed in human epithelial tumour cells, we discovered two members of a novel protein kinase family [Dorow. D. S., Devereux, L., Dietzsch, E. & de Kretser, T. A. (1993) Eul: J. Biochem. 213, 701 -7101. Due to the unique mixture of structural domains within their amino acid sequences, we named the family mixed-lineage kinases (MLK). We initially isolated clones encoding partial cDNAs of MLKl and 2 from a human colonic cDNA library. The MLK2 cDNA was subsequently used to screen a human brain cDNA library and we have now cloned and sequenced a 3454-bp cDNA encoding the full-length MLK2 protein. The predicted MLK2 polypeptide has 954 amino acids and contains a src homology 3 (SH3) domain, a kinase catalytic domain, a double leucine zipper and basic domain, and a large Cterminal domain. The 22-amino-acid N-terminal region has four glutamic acid residues immediately following the initiator methionine. Beginning at amino acid 23, the 55-amino-acid SH3 domain contains a 5-amino-acid insert in a position corresponding to inserts of 6 and 15 residues in the SH3 domains of n-src and the phosphatidylinositol -7'-kinase. Adjacent to the SH3 domain is a kjnase catalytic domain with conserved motifs associated with both serinehhreonine and tyrosine specificity. Beginning nine residues C-terminal to the catalytic domain, there are two Icucine/isoleucine zippers separated by a 13-aminoacid spacer sequence and followed by a stretch of hasic residues. Thc polybasic sequence contains a motif that is similar to nuclear localisation signals from several proteins. The C-terminal domain is composed of 491 amino acids of which 17% are serine or threonine and l h % are proline. This domain also has a biascd ratio of basic to acidic amino acids with ii calculated pl of 9.38. When used as a probe to examine mRNA expression in human tissues, ii MLK2 cDNA hybridised to a species of 3.8 kb that was expressed at highest levels in RNA from brain and skeletal muscle. The 3454-bp cDNA was also used for fluorescence in situ hybridisation to localise the MLR2 gene to human chromosome 19 q13.2.